Symbiotic nitrogen-fi xing bacterial populations trapped from soils under agroforestry systems in the Western Amazon

Cowpea (Vigna unguiculata) is an important grain-producing legume that can forego nitrogen fertilization by establishing an effi cient symbiosis with nitrogen-fi xing bacteria. Although inoculating strains have already been selected for this species, little is known about the genotypic and symbiotic diversity of native rhizobia. Recently, Bradyrhizobium has been shown to be the genus most frequently trapped by cowpea in agricultural soils of the Amazon region. We investigated the genetic and symbiotic diversity of 148 bacterial strains with different phenotypic and cultural properties isolated from the nodules of the trap species cowpea, which was inoculated with samples from soils under agroforestry systems from the western Amazon. Sixty non-nodulating strains indicated a high frequency of endophytic strains in the nodules. The 88 authenticated strains had varying symbiotic effi ciency. The SPAD (Soil Plant Analysis Development) index (indirect measurement of chlorophyll content) was more effi cient at evaluating the contribution of symbiotic N2-fi xation than shoot dry matter under axenic conditions. Cowpea-nodulating bacteria exhibited a high level of genetic diversity, with 68 genotypes identifi ed by BOX-PCR. Sequencing of the 16S rRNA gene showed a predominance of the genus Bradyrhizobium, which accounted for 70 % of all strains sequenced. Other genera identifi ed were Rhizobium, Ochrobactrum, Paenibacillus, Bosea, Bacillus, Enterobacter, and Stenotrophomonas. These results support the promiscuity of cowpea and demonstrate the high genetic and symbiotic diversity of rhizobia in soils under agroforestry systems, with some strains exhibiting potential for use as inoculants. The predominance of Bradyrhizobium in land uses with different plant communities and soil characteristics refl ects the adaptation of this genus to the Amazon region.


Introduction
Agroecosystems generally support lower plant species diversity than ecosystems without human intervention.The reduction in above-ground diversity can have a marked infl uence on the diversity of edaphic organisms, affecting the biochemical processes essential for the sustainability of the forest.Agroforestry systems that seek to minimize these effects have been implemented by many communities in the Amazon, including those of the upper Solimões River (http://www.biosbrasil.ufla. br/).Several researchers conducted studies in the Amazon biome and have demonstrated the great diversity of N 2 -fi xing legume-nodulating bacteria (LNB) strains isolated from nodules in the fi eld or by using different species as trap plants under controlled conditions to capture LNB from the soil (Guimarães et al., 2012;Chagas Junior et al., 2010;Lima et al., 2009;Moreira et al., 1998;Moreira et al., 1993).
Cowpea [Vigna unguiculata (L.) Walp] is an important food crop in Africa and Asia, and the most widely grown grain legume in northern and northeast Brazil, where it is grown primarily by small producers and represents one of the main subsistence crops.This species is considered promiscuous and can establish symbiosis with several species and genera of LNB belonging to the alphaproteobacteria and betaproteobacteria classes (Moreira, 2006).Nevertheless, cowpea can respond to inoculation with select strains of the genus Bradyrhizobium with an increase in productivity (Guimarães et al., 2012).The use of cowpea in studies assessing LNB diversity in soils is relevant because it also allows for the identifi cation of native strains that are adapted to local conditions and potentially useful as inoculants.These studies also assess the need for inoculation when efficient native strains are not available.
Recently, Bradyrhizobium was shown to be the predominant microsymbiont of cowpea in agricultural soils from the Amazon (Guimarães et al., 2012).Agroforestry systems in the same region have varying soil characteristics (Moreira et al., 2009) as well as varying diversity of plant species, which could affect rhizobia diversity.The objective of this study was to investigate the genetic and symbiotic diversity of nitrogen-fi xing bacteria from agroforestry systems in the upper Solimões River, western Amazon.Additionally, the indirect measurement of

Strain authentication and symbiotic effi ciency
Authentication as symbiotic nitrogen-fi xing bacteria was verifi ed for all 148 strains via procedures described in detail by Guimarães et al. (2012), in two successive experiments carried out in axenic conditions with nutrient solution (Hoagland and Arnon, 1950) in the greenhouse.The experiments were conducted over a period of 35 days, and 75 and 73 strains were analyzed in the fi rst and second experiments, respectively.Strains approved as inoculants to cowpea by the Brazilian Ministry of Agriculture were included as positive controls (Moreira, 2006;Martins et al., 2003).
Symbiotic effi ciency of nitrogen-fi xing bacteria was also evaluated with the SPAD (Soil Plant Analysis Development) index of leaves of the middle and upper thirds of the plant, determined using a Minolta SPAD-502 chlorophyll meter that was previously calibrated according to the manufacturer's instructions by averaging 30 readings per plant.The indirect measurement of chlorophyll content using Minolta SPAD-502 chlorophyll meters has been validated for crops such as maize, beans, and soybean, demonstrating a positive, linear correlation between SPAD index values and the nitrogen content of plants and other related variables (Pan and Smith, 2000;Argenta et al., 2002;Poustini et al., 2007;Remans et al., 2008;Vollmann et al., 2011).
All data were tested for normality.The results were analyzed by analysis of variance (ANOVA) with the number of nodules (NN) transformed to the square root of (x + 1) as recommended by SAS Learning Edition 2.0.Mean values were grouped by the Scott-Knott test (p  0.05) using SISVAR (Ferreira, 2011).
The genetic diversity of the strains was analyzed by the presence or absence of polymorphic bands in the gel.The data were grouped with the Unweighted Pair Group Mean Arithmetic Method (UPGMA) algorithm and Jaccard coeffi cient using BioNumerics 6.5 software (Applied Maths, Sint-Martens-Latem, Belgium).

Characterization of genetic diversity by sequencing of the 16S rRNA gene
A total of 22 strains, including at least one from each cultural group and representative of genotypes determined by BOX-PCR, were selected for sequencing of the 16S rRNA gene.Bacterial growth conditions, extraction of genomic DNA (Ausubel et al., 2005), DNA quantifi cation, and PCR conditions (Lane, 1991) are described in Guimarães et al. (2012).
The quality of sequences was verifi ed using Phred and submitted to BLAST for comparison with other se-and without nitrogen supplementation (52.5 mg N L -1 ) showed no nodulation, indicating no external contamination in the experiment, which is a requirement for this type of study.
No strain had shoot dry matter (SDM) similar to the control with high nitrogen content [corresponding to the highest relative effi ciency (RE); statistical "group a"].Strains exhibited varying REs and were grouped by the Scott-Knott test into two groups: intermediate (statistical group "b"), which comprised 12 strains, and ineffi cient (statistical group "c"), which comprised 80 strains and the control without nitrogen supplementation.Among the reference strains, which have been used as cowpea inoculants, the BR 3267 strain (RE = 56 %) performed best.Among the land use system strains, UFLA 03-282 (RE = 79 %), which was identifi ed as Rhizobium etli with 99 % similarity (Table 1), demonstrated the highest effi ciency.
According to the SPAD index, the reference strains UFLA 03-84, INPA 03-11B, and BR 3267, a group of 51 strains, and the control supplemented with nitrogen demonstrated the highest values (statistical group "a"), indicating that these strains were effective at fi xing nitrogen, which was not refl ected in the SDM.The other 16 strains were considered ineffi cient (statistical group "c"), as they were similar to the control without N supplementation.The remaining 21 strains had intermediate effi ciency (statistical group "b").Signifi cant and positive correlation coeffi cients were obtained for SPAD index with both SDM (r = 0.47, p < 0.01) and relative effi ciency (r = 0.52, p < 0.01).
A total of 75 genotypes, most of which were restricted to only one strain, were obtained after clustering the BOX-PCR profi les with over 70 % similarity (Figure 2).The 73 strains from the agroforestry system, which were distributed among 68 genotypes, did not exhibit a banding pattern similar to any of the seven reference/ type strains evaluated, indicating a high level of diversity among them.
After genotypes were determined by BOX-PCR, 23 strains were selected for phylogenetic analysis and partial sequencing of the region 3´ -5´ that encodes the 16S rRNA gene.These sequences ranged between 416 bp and 850 bp and exhibited 96-100 % identity with existing sequences in GenBank (Table 1).The phylogenetic relationships between the sequences are shown in Figure 3.
Sequencing of the 16S rRNA gene indicated that all sampling sites exhibited great diversity (Table 1).The largest number of strains (70 %) belonged to the genus Bradyrhizobium, and most of these were Bradyrhizobium sp. at varying percentages of similarity.Other genera identifi ed were: Rhizobium, Ochrobactrum, Paenibacillus, Bosea, Bacillus, Enterobacter, and Stenotrophomonas.

Discussion
The fact that non-nodulating strains were predominantly fast-growing indicates that these strains were in-quences deposited in GenBank (http://www.ncbi.nlm.nih.gov/GenBank).Only sequences greater than 400 bp in length were used in the phylogenetic analysis.Sequence alignment was performed with ClustalW, and the phylogenetic tree was constructed using the neighborjoining method in the Kimura 2 model (Krasova-Wade et al., 2003;Saitou and Nei, 1987) using the parameters in MEGA version 5 (Tamura et al., 2011).A bootstrap confidence analysis was performed with 1000 replications.

Nucleotide sequence accession numbers
The sequences determined in this work have been deposited in GenBank under accession numbers KC113601 to KC113623.
Of the 148 strains evaluated in the two authentication experiments, 88 (59 %) exhibited a capacity to nodulate.Strains that failed to nodulate were predominantly fast-growing, and among these, the most frequent were those that acidifi ed the medium (FA).Controls with ternal or external contaminants of the nodules that grew faster than the rhizobia during the isolation process because nodules were fi rm, exhibited no signs of decomposition, and were harvested from fully-grown plants that were not in senescence.
The SPAD index, an indirect measurement of chlorophyll content, confi rmed the N 2 effi ciency of various strains that presented the highest SDM, and the SPAD index and SDM were signifi cantly correlated.However, while a total of 51 strains (out of 88) were considered effi cient by the SPAD index (because they were similar to the control with mineral nitrogen), none reached the SDM of the mineral nitrogen.This occurred probably due to the short period plants were allow to grow (35 days), during which nitrogen uptake was not yet converted to dry matter.Thus, although the measurement of SDM per se is fast and less expensive, SPAD is a more effective method for evaluating symbiotic N 2 fi xation under axenic conditions over shorter periods.On the other hand, the equipment to measure the SPAD index is not expensive and can provide a faster analysis than N anal-ysis by Kjeldahl method.These results corroborate results obtained in pots with Phaseolus vulgaris (Poustini et al., 2007).Under fi eld conditions signifi cant correlations between SPAD index and yields were obtained for maize (Argenta et al., 2002), beans (Remans et al., 2008), and soybean (Pan and Smith, 2000;Volmann et al., 2011).
A high diversity of the native rhizobia populations of agroforestry was revealed by rep-PCR, a high-resolution technique used to assess bacterial diversity (Versalovic et al., 1994).Rep-PCR is considered to be a universal tool for studies of genomic variation in prokaryotes and refl ects the variability of the entire genome (Igual et al., 2001).In our study, BOX-PCR, which is a specifi c type of rep-PCR, proved to be an effi cient technique for assessing the diversity of nitrogen-fi xing bacteria that nodulate cowpea from agroforestry systems.The high diversity of strains was confi rmed by 16S rRNA sequence, although 16S rRNA gene sequencing does not offer good species-level resolution among members of the Bradyrhizobium, thus requiring further testing to identify species belonging to this genus (Guimarães et al., 2012;Willems et al., 2001).In addition to Bradyrhizobium spp., other species and genera were identifi ed in this study: Bacillus pumilus, Paenibacillus daejeonensis, Paenibacillus humicus, Enterobacter cloacae, Bosea thiooxidans, Stenotrophomonas maltophillia, Rhizobium etli, and Ochrobactrum anthropi.The latter two species belong to N 2 -fi xing, legume-nodulating bacterial genera, although O. anthropi has not previously been reported as such.Lima et al. (2009) reported that the genus Stenotrophomonas is potentially legume-nodulating in the same study area as in the present study, but there are no reports on the nodulation capacity of the other species, and further work is needed to confi rm that they are indeed LNBs and not non-symbiotic endophytes that are commonly isolated from legume nodules (Sprent, 2009;Gyaneshwar et al., 2011).Their taxonomic position and nodulation capacity should be assessed in future studies.Nevertheless, there are reports that these species promote plant growth through other processes.Silva et al. (2007) studied the effects of different Paenibacillus macerans, Paenibacillus durus, Paenibacillus polymyxa, and B. pumilus strains on the symbiosis between Bradyrhizobium and cowpea, and showed that these strains stimulated nodulation and improved nitrogen fi xation effi ciency.
Few studies in Brazil have assessed the genotypic diversity of native populations that establish symbiotic relationships with cowpea.Zilli et al. (2006) sequenced the 16S rRNA gene from 14 strains: 11 isolated from cowpea nodules of plants grown in Cerrado soils in Piauí state (BR); one isolated from the semi-arid region of Pernambuco state (BR); one isolated from the Integrated Agroecology Production System (Sistema Integrado de Produção Agroecológica -SIPA) in Seropédica, state of Rio de Janeiro, Brazil (22º48'S; 43º41'S, 33 m a.s.l); and one strain (BR2001) previously approved by the Brazilian Ministry of Agriculture (MAPA) as a cowpea inoculant but later replaced by the reference strains cited in this study.Of these 14 strains, nine grouped with Bradyrhizobium elkanii (ATTC49852//USDA 76 T ) with a similarity of 98 %, three with Bradyrhizobium japonicum type strain (ATCC10324 T /LMG 6138) and B. liaoningense (ATCC700350/LMG18230) strains, and two grouped indirectly with B. elkanii and with the group formed by B. japonicum and B. liaoningense.The diversity of 20 rhizobia isolates from upland and fl oodplain soils based on morphological, physiological, and symbiotic characteristics was reported in another study conducted in the Amazon using cowpea as a trap plant (Chagas Junior et al., 2010).However, genetic diversity and identifi cation of rhizobia were not provided in the study.
A few strains, isolated from nodules collected from cowpea growing in the fi eld, were identifi ed as Bradyrhizobium by 16S-23S rDNA IGS sequencing, after screening of a larger number of nodules (88 and 270) by PCR-RFLP analysis of this gene in Senegal, West Africa (Krasova-Wade et al., 2004) (four strains) and South Africa, Ghana, and Botswana (Pule-Meulenberg et al., 2010) (13 strains).It may be that the small number of strains sequenced and bias in obtaining isolates, such as restriction to slow-growing alkalinizing isolates, hindered the isolation of other genera.Lima et al. (2009) identifi ed rhizobia from different land use systems in the western Amazon, including the same agroforestry system sampling points of this study, using siratro (Macroptilium atropurpureum) as a trap plant.The authors identifi ed the following species in the agroforestry system: R. etli, R. tropici, R. galegae, Rhizobium sp., Sinorhizobium medicae, Burkholderia sp., Azorhizobium sp., Bradyrhizobium sp., B. elkanii, B. japonicum, and Mesorhizobium sp.Some, but not all, of these species were also identifi ed in the present study, in addition to other genera (Paenibacillus, Stenotrophomonas, Enterobacter, Bosea, and Ochrobactrum).These results illustrate the need to use more than one species of trap plant when assessing the diversity of nitrogenfi xing, legume-nodulating bacteria in native populations; moreover, siratro captures a higher diversity of legumenodulating bacteria than cowpea.Guimarães et al. (2012) found high genetic and symbiotic diversity of strains isolated from agricultural soils in the same sampling area by using cowpea as trap plant.Although they also found Bradyrhizobium to be a predominant genus among isolates, other genera were found (Rhizobium, Burkolderia, and Achromobacter).However, a lower diversity was found when compared to agroforestry systems, probably due to the higher diversity of legume genera in agroforestry systems (15) than in agricultural systems (6).Thus, the diversity of phenotypes, genotypes, and species of legume-nodulating bacteria found in this study is probably associated with the diversity of legumes in this land use system, because they belong to genera that have already been reported to host a great diversity of legume-nodulating bacteria (Moreira, 2006).Even with the more diverse plant composition and soil attributes in agroforestry than in agriculture, the genus Bradyrhizobium was the most frequent trapped by cowpea, although this species is also able to nodulate with many other genera like Burkholderia, Azorhizobium, Mesorhizobium, Sinorhizobium, and Rhizobium (Moreira, 2006), Achromobacter (Guimarães et al., 2012), and Cupriavidus (Silva et al., 2012).
In conclusion, cowpea was found to nodulate with a high diversity of nitrogen-fi xing bacteria genotypes isolated from soils under agroforestry systems.Most genotypes (70 %) were identifi ed as belonging to different species in the genus Bradyrhizobium, while the remaining genotypes belonged to the genera Rhizobium, Ochrobactrum, Stenotrophomonas, Paenibacillus, Enterobacter, and Bosea.The nodulating strains exhibited varying N 2 -fi xing effi ciencies, but most of them established an effi cient symbiosis with cowpea.The SPAD index was more effi cient than SDM at evaluating the contribution of symbiotic N 2 -fi xation under axenic conditions over shorter periods.

Figure 1 -
Figure 1 -Distribution in cultural groups of 148 bacterial strains isolated from soil samples under agroforestry systems according to growth rate and alteration of medium pH: fast-growing with medium acidifi cation (FA), no alteration of pH (FN), or medium alkalinization (FAL); intermediate-growing with medium acidifi cation (IA), no alteration of pH (IN), or medium alkalinization (IAL); slowgrowing with medium acidifi cation (SA), no alteration of pH (SN), or medium alkalinization (SAL).

aFigure 2 -Figure 3 -
Figure 2 -Dendrogram based on profi les obtained by rep-PCR using the BOX primer of cowpea-nodulating strains isolated from soil samples under agroforestry systems in the western Amazon and of type strains.Genotypes were determined using similarity values of 70 %.*indicates the isolates for which 16S rRNA gene has been sequenced (Table1).

Table 1 -
Origin (sampling point), relative effi ciency, SPAD index, cultural characteristics and identifi cation (based on 16S rRNA sequences extant in GenBank) of strains that nodulate and fi x nitrogen in symbiosis with cowpea.